Litopenaeus vannamei;, animal by-products;, digestive enzymes;, metagenomics;, nutrition.


This study aimed to evaluate the use of protein hydrolysate of poultry by-product and swine liver in the diet of Litopenaeus vannamei and its effect on the intestinal microbiota and on the enzymatic activity of the hepatopancreas. Shrimp (10.94 ± 0.90 g) were fed with diets containing 0%, 25%, 50%, 75% and 100% of replacement of salmon by-product meal by protein hydrolysate, in triplicate. The hepatopancreas enzymatic activity and composition of intestinal microbiota was studied. It was observed that the protein hydrolysate in the diet changed the enzymatic activity of the shrimp when compared to the control group (p < 0.05). Amylase activity increases directly with the percent of protein replacement in the diet. Metagenomic analysis revealed change in the gut biome of the shrimps. The increasing levels of protein replacement provided greater richness in the 75% and 100% treatments, were mainly related to changes in the abundances in the families Rhodobacteraceae and Flavobacteriaceae. A reduction in the abundance of the Vibrionaceae family was observed with the inclusion of protein hydrolysate in the diet. These results indicate that the protein hydrolysate demonstrated beneficial changes when added at concentrations of 25% in the diet of L. vannamei.


Abell, G.C.J.; Bowman, J.P. 2005. Ecological and biogeographic relationships of class Flavobacteria in the Southern Ocean. FEMS Microbiology Ecology, 51(2): 265-277.

Alvarez-González, C.A. 2003. Actividad enzimática digestiva y evaluación de dietas para el destete de larvas de la cabrilla arenera Paralabrax maculatofasciatus (Percoidei:Serranidae), Baja California Sur, México, La paz. 166f (Doctoral thesis. Instituto Politécnico Nacional). Available at: <> Accessed: Feb. 12, 2019.

Caporaso, J.; Lauber, C.; Walters, W.; Berg-Lyons, D.; Lozupone, C.; Turnbaugh, P.; Fierer, N.; Knight, R. 2011. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. National Academy of Sciences, 108(2): 4516-4522.

Castro, R.J.S.; Inacio, R.F.; Oliveira, A.L.R.; Sato, H.H. 2016. Statistical optimization of protein hydrolysis using mixture design: Development of efficient systems for suppression of lipid accumulation in 3T3-L1 adipocytes. Biocatalysis and Agricultural Biotechnology, 5(1): 17-23.

Chakka, A.K.; Elias, M.; Jini, R.; Sakhare, P.Z.; Bhaskar, N. 2015. In-vitro antioxidant and antibacterial properties of fermentatively and enzymatically prepared chicken liver protein hydrolysates. Journal of Food Science and Technology, 52(12): 8059-8067.

Chalamaiah, M.; Kumar, B.D.; Hemalatha, R.; Jyothirmayi, T. 2012. Fish protein hydrolysates: Proximate composition, amino acid composition, antioxidant activities and applications. Food Chemistry, 135(4): 3020-3038.

Cordova-Murueta, J.; Garcí­­a-Carreí­±o, F. 2002. Nutritive value of squid and hydrolyzed protein supplement in shrimp feed. Aquaculture (Amsterdam, Netherlands), 210(4): 371-384.

Cordova-Murueta, J.H.; Garcia-Carreí­±o, F.L.; Navarrete-Del-Toro, M. 2003. Digestive enzymes present in crustacean feces as a tool for biochemical, physiological, and ecological studies. Journal of Experimental Marine Biology and Ecology, 297(1): 43-56.

Erlanger, B.F.; Kokowsky, N.; Cohen, W. 1961. The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics, 95(2): 271-278.

Gamboa-Delgado, J.; Molina-Poveda, C.; Cahu, C. 2003. Digestive enzyme activity and food ingesta in juvenile shrimp Litopenaeus vannamei (Boone, 1931) as a function of body weight. Aquaculture Research, 34(15): 1403-1411.

Gao, S.; Pan, L.; Huang, F.; Song, M.; Tian, M.; Zhang, M. 2019. Metagenomic insights into the structure and function of intestinal microbiota of the farmed Pacific white shrimp (Litopenaeus vannamei). Aquaculture, 499(2): 109-118.

Garcí­­a-Carreí­±o, F.L.; Dimes, L.E.; Haard, N.F. 1993. Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Analitycal Biochemistry, 214(1): 65-69.

Geiger, R.; Fritz, H. 1988. Trypsin. In: Bergmeyer, H.U.; Grab, M. (eds). Methods of enzymatic analysis. New York: Academia Press, p. 119-129.

Hernández, C.; Olvera-Novoa, M.A.; Smith, D.M.; Hardy, R.W.; Gonzalez-Rodriguez, B. 2011. Enhancement of shrimp Litopenaeus vannamei diets based on terrestrial protein sources via the inclusion of tuna by-product protein hydrolysates. Aquaculture, 317(1): 117-123.

Hernandez-Cortés, P.; Whitaker, J.R.; Garcí­­a-Carreí­±o, F.L. 1997. Purification and characterization of chymotrypsin from Penaeus vannamei (Crustacean: Decapoda). Journal of Food Biochemistry, 21(1): 497-514.

Hjelm, M.; Riaza, A.; Formoso, F.; Melchiorsen, J.; Gram, L. 2004. Seasonal incidence of autochthonous antagonistic Roseobacter spp. and Vibrionaceae strains in a turbot larva (Scophthalmus maximus) rearing system. Applied and Environmental Microbiology, 70(12): 7288-7294.

Hou, Y.; Wu, Z.; Dai, Z.; Wang, G.; Wu, G. 2017. Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance. Journal of Animal Science and Biotechnology, 8(1): 8-24.

Izadpanah, A.; Gallo, R.L. 2005. Antimicrobial peptides. Journal of the American Academy of Dermatology, 52(3): 381-390.

Izvekova, G.I. 2006. Hydrolytic activity of enzymes produced by symbiotic microflora and its role in digestion processes of bream and its intestinal parasite Caryophyllaeus laticeps (Cestoda, Caryophyllidea). The Biological Bulletin, 33(3): 287-292.

Kar, N.; Ghosh, K. 2008. Enzyme producing bacteria in the gastrointestinal tracts of Labeo rohita (Hamilton) and Channa punctatus (Bloch). Turkish Journal of Fisheries and Aquatic Sciences, 8(1): 115-120.

Kelly, C.; Salinas, I. 2017. Under pressure: interactions between commensal microbiota and the teleost immune system. Frontiers in Immunology, 8(1): 1-8.

Kim, S.W.; Less, J.F.; Wang, L.; Yan, T.; Kiron, V.; Kaushic, S.J.; Lei, X.G. 2018. Meeting global feed protein demand: challenge, opportunity, and strategy. Annual Review of Animal Biosciences, 7(2): 1-17.

Kirchman, D.L. 2002. The ecology of Cytophaga-Flavobacteria in aquatic environments. FEMS Microbiology Ecology, 39(2): 91-100.

Kristinsson, H.G.; Rasco, B.A. 2000. Fish protein hydrolysates: production, biochemical, and functional properties. Critical Reviews in Food Science and Nutrition, 40(1): 43-81.

Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature, 227(1): 680-685.

Lemos, D.; Ezquerra, J.M.; Garcia-Carreí­±o, F.L. 2000. Protein digestion in penaeid shrimp: digestive proteinases, proteinase inhibitors and feed digestibility. Aquaculture, 186(1): 89-105.

Li, E.; Chang, X.; Wang, X.; Wang, S.; Zhao, Q.; Zhang, M.; Quin, J.; Chen, L. 2018. Gut microbiota and its modulation for healthy farming of pacific white shrimp Litopenaeus vannamei. Reviews in Fisheries Science & Aquaculture, 26(3): 381-399.

Lowry, O.H.; Rosenbrough, N.J.; Farr, A.L.; Randall, R.J. 1951. Protein measurement with the Folin-Phenol reagent. The Journal of Biological Chemistry, 193(3): 265-276.

Marouax, S.; Louvard, D.; Barath, J. 1973. The aminopeptidase from hog intestinal brush border. Biochimica Et Biophysica Acta (BBA) - Enzymology, 321(1): 282-295.

Martí­­nez-Alvarez, O. 2013. Hormone-like peptides obtained by marine-protein hydrolysis and their bioactivities. In: Kim, S.D. (ed). Marine proteins and peptides: Biological activities and applications. Chichester: John Wiley & Sons. p. 351-367.

Mata, M.T.; Luza, M.F.; Riquelme, C.E. 2017. Production of diatom-bacteria biofilm isolated from Seriola lalandi cultures for aquaculture application. Aquaculture Research, 48(8): 4308-4320.

Moullac, G.L.; Klein, B.; Sellos, D.; Wormhoudt, A.V. 1996. Adaptation of trypsin, chymotrypsin and α-amylase to casein level and protein source in Penaeus vannamei (Crustacea Decapoda). Journal of Experimental Marine Biology and Ecology, 208(1): 107-125.

Muhlia-Almazán, A.; Garcí­­a-Carreí­±o, F.L. 2002. Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 133(3): 383-394.

Nasri, M. 2016. Protein hydrolysates and biopeptides: production, biological activities, and applications in foods and health benefits. A review. Advances in Food and Nutrition Research, 81(3): 109-159.

Nelson, D.L.; Cox, M.M. 2011. Princí­­pios de bioquí­­mica de Lehninger. 5. ed. Porto Alegre: Artmed, 1304 p.

Nguyen, H.T.M.; Pérez-Gálvez, R.; Bergé, J.P. 2012. Effect of diets containing tuna head hydrolysates on the survival and growth of shrimp Penaeus vannamei. Aquaculture, 324(1): 127-134.

Niu, J.; Xie, J.; Guo, T.; Fang, H.; Zhang, Y.; Liao, S.; Xie, S.; Liu, Y.; Tian, L. 2019. Comparison and evaluation of four species of macro-algaes as dietary ingredients in Litopenaeus vannamei under normal rearing and WSSV challenge conditions: effect on growth, immune response, and intestinal microbiota. Frontiers in Physiology, 9(1): 1-15.

Osman, A.; Goda, H.A.; Abdel-Hamid, M.; Badran, S.M.; Otte, J. 2016. Antibacterial peptides generated by alcalase hydrolysis of goat whey. Lebensmittel-Wissenschaft + Technologie, 65(2): 480-486.

Ramirez, R.F.; Dixon, B.A. 2003. Enzyme production by obligate intestinal anaerobic bacteria isolated from oscars (Astronotus ocellatus), angelfish (Pterophyllum scalare) and southern flounder (Paralichthys lethostigma). Aquaculture, 227(3): 417-426.

Restrepo, L.; Bayote, B.; Arciniegas, S.; Bají­±a, L.; Betancourt, L.; Panchana, F.; Munoz, A.R. 2018. PirVP genes causing AHPND identified in a new Vibrio species (Vibrio punensis) within the commensal Orientalis clade. Scientific Reports, 8(1): 1-14.

Rick, W.; Stegbauer, H.P. 1974. Alpha amylase of reducing groups. In: Bergmeyer, H.V. (ed.). Methods of enzymatic analysis. New York: Academic Press, p. 885-890.

Rigottier-Gois, L. 2013. Dysbiosis in inflammatory bowel diseases: the oxygen hypothesis. The ISME Journal, 7(2): 1256-1261.

Rosas, C.; Cuzon, G.; Gaxiola, G.; Arena, L.; Lemaire, L.; Soyez, C.; Wormhoutdt, A.V. 2000. Influence of dietary carbohydrate on the metabolism of juvenile Litopenaeus stylirostris. Journal of Experimental Marine Biology and Ecology, 249(2): 181-198.

Saadi, S.; Saari, N.; Anwar, F.; Hamid, A.A.; Ghazali, H.M. 2015. Recent advances in food biopeptides: Production, biological functionalities and therapeutic applications. Biotechnology Advances, 33(1): 80-116.

Safari, R.; Motamedzadegan, A.; Ovissipour, M.; Regenstein, J.M.; Gildberg, A.; Rasco, B. 2012. Use of hydrolysates from Yellowfin tuna (Thunnus albacares) heads as a complex nitrogen source for lactic acid bacteria. Food and Bioprocess Technology, 5(1): 73-79.

Schippa, S.; Conte, M. 2014. Dysbiotic Events in Gut Microbiota: Impact on Human Health. Nutrients, 6(12): 5786-5805.

Sharifah, E.N.; Eguchi, M. 2011. The Phytoplankton Nannochloropsis oculata enhances the ability of Roseobacter clade bacteria to inhibit the growth of fish pathogen Vibrio anguillarum. PLoS One, 6(10): e26756.

Silchenko, A.S.; Rasin, A.B.; Kusaykin, M.I.; Malyarenko, O.S.; Shevchenko, N.M.; Zueva, A.O.; Kalinovsky, A.I.; Zvyagintseva, T.N.; Ermakova, S.P. 2018. Modification of native fucoidan from Fucus evanescens by recombinant fucoidanase from marine bacteria Formosa algae. Carbohydrate Polymers, 193(1): 189-195.

Silchenko, A.S.; Ustyuzhanina, N.E.; Kusaykin, M.I.; Krylov, V.B.; Shashkov, A.S.; Dmitrenok, A.S.; Usoltseva, R.V.; Zuevab, A.O.; Nifantievc, N.E.; Zvyagintseva, T.N. 2016. Expression and biochemical characterization and substrate specificity of the fucoidanase from Formosa algae. Glycobiology, 27(3): 254-263.

Soares, M.; Rezende, P.C.; Corrêa, N.M.; Rocha, J.S.; Martins, M.A.; Andrade, T.C.; Fracalossi, D.M.; Vieira, F.N. 2020. Protein hydrolysates from poultry by-product and swine liver as an alternative dietary protein source for the Pacific white shrimp. Aquaculture Reports, 17(2): 100344.

Sowmya, R.; Sachindra, N.M. 2015. Carotenoid production by Formosa sp. KMW, marine bacteria of Flavobacteriaceae family: Influence of culture conditions and nutrient composition. Biocatalysis and Agricultural Biotechnology, 4(4): 559-567.

Thompson, F.L.; Iida, T.; Swings, J. 2004. Biodiversity of Vibrios. Microbiology and Molecular Biology Reviews, 68(3): 403-431.

Tzuc, J.T.; Escalante, D.R.; Herrera, R.R.; Cortés, G.G.; Ortiz, M.L.A. 2014. Microbiota from Litopenaeus vannamei: digestive tract microbial community of Pacific white shrimp (Litopenaeus vannamei). SpringerPlus, 3(1): 280-290.

Verma, A.K.; Chatli, M.K.; Kumar, P.; Mehta, N. 2017. Antioxidant and antimicrobial activity of protein hydrolysate extracted from porcine liver. The Indian Journal of Animal Sciences, 87(2): 711-717.

Villamil, O.; Váquiro, H.; Solanilla, J.F. 2017. Fish viscera protein hydrolysates: production, potential applications and functional and bioactive properties. Food Chemistry, 224(1): 160-171.

Villasante, F.; Fernández, I.; Preciado, R.M.; Oliva, M. 1999. The activity of digestive enzymes during the molting stages of the arched swimming Callinectes arcuatus Ordway, 1863 (Crustacea: Decapoda: Portunidae). Bulletin of Marine Science, 65(3): 1-9.

Wald, M.; Schwarz, K.; Rehbein, H.; Buí­Å¸mann, B.; Beermann, C. 2016. Detection of antibacterial activity of an enzymatic hydrolysate generated by processing rainbow trout by-products with trout pepsin. Food Chemistry, 205(1): 221-228.

Wang, Y.; Qian, P.Y. 2009. Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One, 4(10): 1-9.

Wu, S.; Wang, G.; Angert, E.R.; Wang, W.; Li, W.; Zou, H. 2012. Composition, diversity, and origin of the bacterial community in grass carp intestine. PLoS One, 7(2): 1-10.

Xiong, J.; Dai, W.; Li, C. 2016. Advances, challenges, and directions in shrimp disease control: the guidelines from an ecological perspective. Applied Microbiology and Biotechnology, 100(2): 47-54.

Yamazaki, Y.; Meirelles, P.M.; Mino, S.; Suda, W.; Oshima, K.; Hattori, M.; Thompson, F.L.; Sakai, Y.; Sawabe, T.; Sawab, T. 2016. Individual Apostichopus japonicus fecal microbiome reveals a link with polyhydroxybutyrate producers in host growth gaps. Scientific Reports, 6(2): 21631.

Zhang, M.; Sun, Y.; Chen, K.; Yu, N.; Zhou, Z.; Chen, L.; Du, Z.; Li, E. 2014. Characterization of the intestinal microbiota in Pacific white shrimp, Litopenaeus vannamei, fed diets with different lipid sources. Aquaculture, 434(4): 449-455.

Zheng, K.; Liang, M.; Yao, H.; Wang, J.; Chang, Q. 2012. Effect of dietary fish protein hydrolysate on growth, feed utilization and IGF-I levels of Japanese flounder (Paralichthys olivaceus). Aquaculture Nutrition, 18(3): 297-303.

Zhou, XX.; Pan, YJ.; Wang, YB.; Li, WF. 2007. In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides. Journal of Zhejiang University. Science. B., 8(9): 686-692.






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